New Glycosidase Substrates for Droplet-Based Microfluidic Screening Majdi Najah, †,‡ Estelle Mayot, †,‡ I Putu Mahendra-Wijaya, †,‡ Andrew D. Griffiths,* ,†,§ Sylvain Ladame, †,∥ and Antoine Drevelle* ,†,‡ † Institut de Science et d’Inge ́ nierie Supramole ́ culaires (ISIS), Universite ́ de Strasbourg, CNRS UMR 7006, 8 allé e Gaspard Monge, 67083 Strasbourg Cedex, France ‡ Ets J. Soufflet, division Biotechnologies-OSIRIS, quai Sarrail, 10400 Nogent-sur-Seine, France § E ́ cole Supe ́ rieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI ParisTech), 10 rue Vauquelin, 75231 Paris Cedex, France ∥ Department of Bioengineering, Imperial College London, South Kensington Campus, London SW72AZ, United Kingdom * S Supporting Information ABSTRACT: Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet micro- fluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30 °C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-D-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet- based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4−11) and with incubation times of up to 24 h in droplet-based microfluidic systems. D espite recent progress in opening up vast new fossil fuel reserves, environmental concerns linked to the produc- tion of greenhouse gases and side effects of hydraulic fracturing make sustainable production of commodities such as biofuels and other chemicals an attractive proposition. For a sustainable bioprocess, it is preferable to reserve starch for the food/feed industry and to instead use abundant renewable materials rich in cellulose and/or hemicellulose as feedstocks. All conversion technologies of these feedstocks proceed via hydrolysis of the biological polymers into simple sugars (mainly glucose and/or xylose), a sustainable bioprocess that requires efficient glycosidases (EC 3.2.1), most notably cellulases and xylanases. 1 As a result of this, improving the efficacy of known glycosidases or finding new glycosidases, especially enzymes involved in the last steps of degradation releasing monosaccharides, which can be converted into commodities by fermentation process in a “sugars” biorefinery, 2 has become a major challenge for the bioconversion industry. In this regard, an efficient and powerful high-throughput screening system is needed to select suitable enzymes. Screening methods based on in vitro compartmental- ization 3 (IVC) combined with fluorescence-activated cell sorting (FACS) 4−6 have been described for directed evolution of a number of enzymes, including β-glucosidase. 7 This method relies on analysis of single cells compartmentalized in water-in- oil-in-water emulsions. 6 It inspired the development of droplet- based microfluidic systems for ultra-high-throughput screening of enzymes and microorganisms, which allows the production, manipulation, and sorting of highly monodisperse picoliter-size microreactors at high frequencies (kilohertz). 8 This has allowed the directed evolution of horseradish peroxidase displayed on the surface of Saccharomyces cerevisiae 9 and the evolution of sulfatase expressed in Escherichia coli. 10 Droplet-based micro- fluidic systems have also been used to sort mammalian cells, 11 viruses, 12 or even genes expressed in vitro. 13,14 For screening in droplet-based microfluidic systems, fluorogenic substrates based on a fluorescent leaving group are typically used to measure enzymatic activities. 13,15 For instance, coumarin-based substrates have been widely used for Received: July 23, 2013 Accepted: September 6, 2013 Published: September 6, 2013 Article pubs.acs.org/ac © 2013 American Chemical Society 9807 dx.doi.org/10.1021/ac4022709 | Anal. Chem. 2013, 85, 9807−9814